The TRA/TRA-2 complex also regulates the sex-specific splicing of downstream effector genes, doublesex (dsx) and fruitless (fru). In Ceratitis capitata, a Maleness-on the-Y (MoY) gene modulates sex-specifically spliced Cctra pre-mRNA and results in the breakdown of the Cctra autoregulatory loop in males. In this study, the tra-2 and fru genes were characterised in two key pests, Bactrocera dorsalis and B. correcta. The tra-2 genes showed high degrees of conservation among tephritids. The complex gene organisation for each of Bdfru and Bcfru were identified. There are sex-specific and non sex-specific transcripts generated by alternative promoters as found in Drosophila melanogaster and other insects. RNAi knockdown of Bdtra transcripts showed that BdTRA controls the sex-specific splicing of Bddsx and Bdfru pre-mRNAs. Developmental expression analysis shows that multiple splice variants of Bdtra and Bctra RNAs are present before and during cellular blastoderm formation and that the mature sex-specific variants become fixed later in embryogenesis. Furthermore, the BddsxM splice variants are found in early embryos at the beginning of gastulation, but BdfruM does not appear until the larval stage. We proposed that the zygotic tra loop is initiated in both female and male embryos before becoming automatised or abolished by MoY, respectively.Sex determination in tephritid fruit flies involves a signaling cascade of alternatively spliced genes. The Transformer (TRA) and Transformer-2 (TRA-2) complex establishes an autoregulatory loop switching sex-specific splicing of  Sex-determination mechanisms are highly diverse, and they usually occur during early development2. The sex determination of insects is one of the most diverse and well-characterised systems4. Its evolutionarily divergent model constitutes three levels of sex-determining regulators4. The primary signals are variable among insect species. Most of their signaling mechanisms are still unknown, but the signals are relayed via alternative splicing of a conserved binary switch gene. This switch gene, encoded for a splicing regulator, relays similar alternative splicing messages to the highly conserved downstream effectors.Sexual reproduction is a key point for the continuation of multicellular eukaryotes. Males and females evolve into sexual dimorphism through the sexual selection processes5. Several of them are high profile pests, including Ceratitis capitata (Mediterranean fruit fly), Bactrocera dorsalis , B. oleae (olive fruit fly), B. tryoni (Queensland fruit fly), Anastrapha ludens (Mexican fruit fly), A. obliqua (West Indian fruit fly), and A. suspensa (Caribbean fruit fly). Many sex-determining orthologues have been characterised to understand the developmental cascades which are also beneficial for determining new pest control strategies8. For instance, genetic sexing strains have been developed by employing modern genetic approaches for pest control programs using the sterile insect technique (SIT)13.Tephritid fruit flies are in a very diverse dipteran family comprising at least 5,000 speciesMaleness-on the-Y (MoY) has been discovered in C. capitata14. The MoY orthologues were also characterised in many Bactrocera spp. such as B. dorsalis, B. oleae, and B. tryoni. Disruption or overexpression of CcMoY can feminise XY embryos or masculinise XX embryos, respectively. CcMoY modulates sex-specific splicing of transformer (tra) pre-mRNA14. In tephritid female flies, the tra and transformer-2 (tra-2) orthologues encode the TRA and TRA-2 proteins that later form a TRA/TRA-2 complex to maintain an autoregulatory loop of the female-specifically spliced tra transcript (traF)25. The Bdtra24 and Bdtra-223 were previously characterised in B. dorsalis. The RNAi-mediated knock-down of any one of these genes generated only males because the autoregulatory loop was disrupted. Likewise, the autoregulatory loop is disrupted by the presence of MoY orthologues in males14. The TRA/TRA-2 complex also switches on the DoublesexF protein (DSXF) by regulating female-specific splicing of dsx pre-mRNA. The switching-off mode results in DSXM because the absence of the functional TRA/TRA-2 complex allows male-specific splicing of dsx pre-mRNA24. DSXF and DSXM are highly conserved functional effectors in insects27. They are transcriptional factors with a masculinised or feminised function for downstream genes, controlling somatic sexual dimorphism28. The dsx orthologues studied in several tephritid fruit flies  are conserved, suggesting similar somatic sexual differentiation among them.Recently, the primary signal fruitless (fru), that confers the sexual behaviors of males38. However, the fru gene has not been characterised in detail among tephritid species. The molecular mechanisms of fru in sex determination were proposed based on non-tephritid models41. The fru genes are very complex loci encoding sex-specific and non sex-specific transcriptional factor isoforms43. FRU proteins generally contain a dimerisation-interface domain called the BTB  domain and one alternative C2H2 zinc finger domain45. Each of the fru orthologues has many putative transcripts derived from any one of the alternative promoter-derived 5′ sex- and non sex-specific exons, which connect to four to five common exons and then connect to any one of the 3′ alternative zinc-finger encoding exons41. Notably, the sex-specifically spliced transcripts are only processed from the most distal promoter (P1) derived pre-mRNA in many non-tephritid species41. In Drosophila melanogaster, the non sex-specific FRU isoforms have developmental functions. They are translated from P2 to P4 transcripts48.In a tephritid and remotely related insects, the TRA/TRA-2 complex switches were also found to control the sex-specific splicing of the other downstream effector gene, tra-2 and fru orthologues in B. dorsalis and B. correcta. The RNAi knockdown experiments showed that BdTRA controls the sex-specific splicing of BdtraM, BddsxM, and BdfruM transcripts in pseudomales. In addition, a comprehensive sex-determination cascade was investigated from the developmental expression profiles of MoY, tra, tra-2, dsx, and fru in single embryos, larvae, pupae, and adults. These results likely ascertained the time frames of critical sex-determining events in these Bactrocera fruit flies.In this study, we report the gene structure and expression of B. dorsalis tra-2 (Bdtra-2) and B. correcta tra-2 (Bctra-2) are approximately 1.2 kilobases (kb) and 1.4 kb, respectively, in both sexes. The genomic DNA and cDNA sequences of Bdtra-2  and Bctra-2  were acquired from adult males and females. Both genes consist of eight exons , B. tryoni21, B. jarvisi21, Z. cucurbiate25, C. capitata18, Anastrepha spp.20, and D. melanogaster50. The open reading frames (ORF) of Bdtra-2 and Bctra-2 are 753 bp, encoding for a predicted polypeptide of 251 amino acids with 98% identity   and B. correcta fru (Bcfru)  genes. Both orthologues consist of three components  to analyse the gene organisation. The results suggested that the Bdfru gene structure is very large in size, spanning approximately 330 kb of genomic sequence  and untranslated exons ) were found and spliced to the middle common exons in four different patterns   exon and its cis-regulatory elements from the genomic scaffold. The 1.9 kb region downstream of the M exon was available in the B. dorsalis genomic scaffold (GenBank Acc. No. NW_011876307.1). The putative TRA/TRA-2 binding sites and multiple in-frame stop codons were in silico identified in this region  of B. dorsalis was used to identify adult pseudomales   Fig. . Male-sples Fig. . Bdtra-2tes Fig. . The resB. dorsalis and B. correcta embryos were very similar when observed from 0 to 9 h after egg laying (h AEL)  were studied in unfertilised eggs and during early embryonic development (1 to 12 h AEL) as well as in larval, pupal, and adult stages  and Bddsx (BddsxF), and Bdtra-2 transcripts in unfertilised eggs shows that these transcripts are contributed maternally. The continued presence of the transcripts in developing embryos indicates zygotic activation of transcription of these genes. The expression of the MoY transcript was only observed in male samples beginning from 3 h AEL embryos, barely visible in larvae, and disappeared in pupae and adults. The combination of BdtraF, zygotic male-specifically spliced Bdtra transcripts (BdtraM), and their intermediate transcripts were firstly observed in both male and female samples at 5 h AEL. The intermediate transcripts consisted of three possible partial sequences of male-specific exon(s)  appeared at the beginning of gastrulation (8 to 10 h AEL) and was detected at all later stages in males. The sex-specific splicing pattern of the Bddsx gene was established in the larvae, pupae, and adults. The male-specifically spliced Bdfru transcript (BdfruM) derived from the P1 promoter was observed in male samples from larval, pupal, and adult stages, but not in the embryonic stage. Notably, a non-specific RT-PCR amplicon was observed and sequenced. It was found to be against predicted B. dorsalis protein peanut and E3 ubiquitin-protein ligase genes in embryos when BLAST was used. The expression profiles of the other Bdfru transcripts derived from the P2, P3, and P4 promoters were also absent in the unfertilised eggs and embryos when tested in 4, 8, and 12 h AEL embryos . This suggests the strong adaptive divergence of sexual behaviors and may confer male-specific transcriptional regulatory roles of FRUM in different insect taxa.The overall deduced amino acid sequences of the BdFRU and BcFRU are generally conserved, as in Bdfru and Bcfru are sex-specifically spliced at the 5′ exons as in the case of D. melanogaster36, An. gambiae39, Ae. Aegypti41, M. domestica38, and N. vitripennis40. The male- and female-specific exons are adjacent. TRA/TRA-2 binding sites located in the female-specific exon suggested an alternative splicing mechanism which selected the 5′ splice site located at the end of the female-specific exon. The binding of TRA/TRA-2 complex hinders the formation of the spliceosome complex at the male-specific splice site in D. melanogaster37. The female-specific FRU protein was truncated because of its in-frame stop codons in the female-specific exon46. Transcripts connecting the female-specific exon and the common C1 exon could not be confirmed by RT-PCR experiments because the female-specifically spliced transcript may be expressed at a low level. It was found that the expression level of the fruF transcripts were lower than the fruM transcripts in D. melanogaster46 and Ae. Aegypti41.The P1 derived primary transcripts of Bdfru and Bddsx are regulated by Bdtra in the sex-determination pathway as previously proposed in the Medfly18. This is based on the presence of TRA/TRA-2 binding sites in their female-specific exons33 (in this study) and the sex-determining gene splicing analysis in the pseudomales made from Bdtra RNAi. The same regulatory roles of Bctra to the Bcfru and Bcdsx genes can be implicated based on the conserved sequences and gene structures33 .The expression profiles of sex-determining genes in Bdtra and Bctra genes were firstly indicated by the appearance of the BdtraM and BctraM transcripts at 5 h AEL for both male and female embryos. However, these transcripts were found in heterogeneity, i.e., all together with BdtraF and BctraF and their intermediate transcripts. The heterogeneous pattern of RNA splicing was equally extended from 5 to 8 h AEL in both sexes. This finding is consistent with the expression pattern of Cctra during the same developmental period55. It is hypothesised that the activation of the zygotic tra loop occurred in all embryos during this period. For the XX embryos, the accumulation of TRA/TRA-2 complex level above a certain threshold would generate a tra-autoregulatory loop. TRA and TRA-2 are serine/arginine (SR) type splicing regulators which operate under threshold dependency56. The presence of BdtraF and BctraF and the absence of BdtraM, BctraM, and their intermediate transcripts suggested that the level of TRA/TRA-2 complex was high enough to complete the automatisation of the tra loop from the 9 h AEL developmental point onward in females. In the case of the XY embryos, the non-autonomous zygotic tra loop was later disrupted by the suppression of the MoY gene. The appearance of mostly BdtraM and BctraM transcripts suggested the breakdown of the tra loop in males beginning at 9 h AEL. This finding is supported by a new discovery of an autosomal derived microRNA, miRNA-1-3p, that is required for male determination of B. dorsalis57. The miRNA-1-3p is involved in the suppression of Bdtra transcripts by targeting 3′ UTR. The relative expression of this miRNA-1-3p is also significantly increased and male-biased during the same critical period of the Bdtra loop breakdown process, i.e., from 6 to 9 h post oviposition at 28 °C. The microRNA is an example of intermediate male determiners. Further bioinformatics and molecular studies that shed light on how MoY relays the instruction to disrupt the tra loop will be focused on the period of 5 to 9 h AEL in B. dorsalis and B. correcta embryos.The expressions of zygotic Bddsx and Bcdsx genes are maternally transcribed. The BddsxF knockdown experiment resulted in an interruption of yolk protein (yp) expression in adults and led to a significant reduction of ovary size, number of oocytes, and malformation of reproductive organs32. The presence of BddsxF and BcdsxF transcripts were observed in both sex embryos up to at least 12 h AEL. DSXF may be used in the expression of genes related to the nourishment of embryos such as the yp gene58. A similar finding showed that the BjdsxF transcript was also expressed in both sex embryos at sometime between 1 to 9 h AEL21. In the medfly, the CcdsxF transcription was maternally derived and also detected at 4 h AEL in all embryos. However, the CcdsxF disappeared from 5 to 9 h AEL and reappeared as a pattern of sex-specifically spliced CcdsxM and CcdsxF transcripts in male and female embryos, respectively, at 10 h AEL55. This indicated that the zygotic expression of the Ccdsx gene had begun around this time. The first zygotic expression of Bddsx and Bcdsx genes were also in evidence at 8 and 9 h AEL, respectively, when the BddsxM and BcdsxM transcripts were detected. In B. jarvisi, a high number of dsxM transcripts was detected after 6 h AEL21.The BddsxF and BcdsxF transcripts were zygotically expressed in male embryos during the period of 9 to 12 h AEL, it would result from the binding between TRA/TRA-2 complex proteins and the dsx pre-mRNA. However, there is evidence that the availability of functional TRA/TRA-2 complex protein is not enough to generate BdtraF and BctraF transcripts from their pre-mRNAs for the same duration. This suggests that TRA/TRA-2 binding complexity at the respective pre-mRNAs of the tra and dsx orthologues are different. Ruiz et al.17 proposed, due to the putative cis-regulatory elements on sex-specific exons of tra and dsx pre-mRNAs, that TRA plays a dual role in the sex-determination pathway of tephritids. The putative TRA/TRA-2 complex binding sites on the female-specific exon of dsx pre-mRNA suggested the requirement of simpler components for the formation of an enhancing complex. In contrast, additional TRA-2 and SR protein(s) may be required for binding at the putative silencing elements on the male-specific exon(s) of tra pre-mRNA17.If the presence of BddsxM and BcdsxM transcripts in cellular blastoderm embryos , Bddsx33 and Bdfru in B. dorsalis. The developmental expression profile analyses can outline the following critical time points. The presence of maternal Bdtra transcripts could provide BdTRA proteins to initiate the autoregulatory loop. The Bdtra-2 transcript is continuously expressed throughout development as found in a previous study22. The Y-linked male-determiner, BdMoY, starts expressing instructions from 3 h AEL until the larval stage. The zygotic tra loop activation becomes apparant in both sex embryos at 5 h AEL. The first male-specific splicing pattern of Bdtra suggests that the BdMOY protein has completed the process of Bdtra loop disruption at 9 h AEL. In addition, the Bdtra autoregulatory loop is fully functional due to the disappearance of BdtraM and intermediate transcripts from 8 to 9 h AEL in female embryos. When the binary Bdtra is switched OFF, it transduces the first signal to the BddsxM effector at 8 h AEL although the appearance of the BddsxF transcript is present in male embryos until at least 12 h AEL. The expression of the BdFRUM effector is not apparent in embryos but only in later development. The comparative studies of all sex-determining orthologues from B. correcta imply similar sex-determination schemes33 (in this study). Detailed study of sex-determination cascades can be focused on more specific times in embryonic and sexual development of tephritid fruit flies. Fundamental mechanisms  can be explained with better resolution. Novel intermediates and accessory proteins required for sex-determination can be screened and validated using bioinformatics and genome editing approaches. Comprehensive understanding of sex-determination may lead to new genetic tools and the improvement of insect pest control strategies in the high profile key pests, B. dorsalis and B. correcta63.The molecular study of B. dorsalis (Phayathai1 strain) and B. correcta (Phayathai2 strain), were used for the isolation of fru and tra-2 genes and the expression analyses of sex-determining genes in different developmental stages. The B. dorsalis Salaya1 genetic sexing strain was used for the RNAi experiment of Bdtra. This strain has sexual phenotypes of brown/white pupae, with males and females emerging from brown and white pupae, respectively64. The fruit flies were reared at 25 °C, with 13 L: 11 D cycles, and around 70% relative humidity (RH) at Regional R&D Training Center for Insect Biotechnology, Mahidol University.The laboratory strains, Two different fruit fly cages were set up for harvesting unfertilised eggs and embryos from sexually mature (14-day old) virgin and mated females, respectively. The induction of egg oviposition was done by putting a pretested oviposition chamber containing guava juice into the fruit fly cages for one hour. The experimental batches of eggs and embryos were harvested from each new oviposition chamber after being left in the cage for 10 min. All samples were incubated at 25 °C and 70% RH. The first batch of eggs was incubated for 48 h to validate virginity and fertilisation of the females from embryo hatching. A series of egg batches were subsequently and independently collected. Each of these egg batches was incubated according to only one specific time point .Collected embryos were dechorionated in 3% sodium hypochlorite solution and washed in DEPC-treated water. Single embryos were individually transferred to a microcentrifuge tube (1.5 ml) before immediate grinding in 100 μl TRIzol reagent . The homogenates were kept at − 80 °C until the RNA isolation step.65. Total RNA was isolated from 2-day old flies using TRIzol reagent according to the manufacturer′s instructions.Genomic DNA was individually extracted according to Baruffi et al.21. The RNA precipitation required an additional 10 µg of RNase-free glycogen  for single embryos. The resuspended RNA solution was evaluated using NanoDrop spectrophotometer .Parallel RNA and genomic DNA extractions were carried out when using single embryos, larvae and pupae. Total RNA was isolated from each sample using TRIzol reagent as per the instructions from the manufacturer. During the RNA isolation, genomic DNA was extracted from the interphase and the organic phase of the TRIzol/chloroform mixB. dorsalis and B. correcta tra-2 genes, the primers were designed from the conserved CDS of Bactrocera spp. and C. capitata tra-2 genes  according to the protocol of the manufacturer. One-tenth of 5′- and 3′-RACE-Ready cDNA samples were subsequently used as templates for RT-PCR and 5′ and 3′ RACEs with gene-specific primers. Semi-nested RT-PCRs were performed to derive a more specific amplification according to SMARTer RACE 5′/3′ Kit. The negative controls were also performed by excluding reverse transcriptase. The tra-2 gene structures were assembled based on PCR experiments using three pairs of PCR primers that were derived from Bdtra-2 and Bctra-2 full-transcripts and genomic DNA templates. The long Bdtra-2 and Bctra-2 cDNA and genomic DNA sequences were deposited to GenBank  exon-encoded sequences were identified from the gene-specific RT-PCR using primers derived from the BTB coding sequence and its 3′ adjacent . Intron/exon junctions of all isolated Bdfru transcripts were analysed by blasting them to the available B. dorsalis genomic scaffold (GenBank Acc. No. NW_011876307). The cDNA sequences of long Bdfru and Bcfru transcripts were deposited to GenBank  gene-specific PCR was used to control false negative samples. Up to 300 ng of total RNA from single embryos, larvae, pupae, and adults were reverse transcribed into the first strand cDNA using ImProm-II Reverse Transcriptase , following the manufacture′s instructions. One-tenth of the initial reverse transcription reaction was used as a template for RT-PCR using gene-specific primers. For each sample, the negative control of RT-PCR was performed by excluding the reverse transcriptase. The gapdh positive control system was also carried out in the RT-PCR. Slam, a zygotically expressed cellularisation gene, was used as an early developmental marker55. Therefore, the 2 to 3 h AEL embryos or older without detectable slam were discarded. Relative locations of gene-specific primers to each sex-determining gene were schematically plotted in Supplementary Table Sexual identification of single embryo, larval, and pupal samples were molecularly identified by genomic PCR using Y-specific primers which were designed based on sequences such as the Bdtra gene was synthesised by in vitro transcription with T7 RNA polymerase using the MEGAscript Kit . Embryos were prepared from the B. dorsalis Salaya1 genetic sexing strain, whose sexual phenotypes can be separated by pupal colour dimorphism64. One μg/μl of Bdtra dsRNA was microinjected at the posterior end of precellular blastoderm embryos. The RNAi-treated individuals were reared until adult emergence. Wild-type male and female adults emerged from brown and white pupae, respectively. The white-pupae males were therefore categorised as pseudomales24. The genitalia and testes of the wild-type males and pseudomales were compared under a microscope. The sex-specific splicing pattern of Bdtra, Bdtra-2, Bddsx, and Bdfru were carried out using the previously described RT-PCR conditions in two pseudomales (randomly selected). The sex-specific splicing patterns of these genes were compared to the wild-type males and females as well as the RNAi-treated wild-type males.Approximately 1.1 kb dsRNA of the E. coli (DH5α) competent cells and screened for positive colonies. Plasmids were extracted using the GF-1 Plasmid DNA Extraction Kit . All sequencing reactions were performed on both strands using the sequencing service from Macrogen Inc., Seoul, Korea.The specific amplicon was purified with the GF-1 AmbiClean Kit . The products were subsequently cloned into the pGEM-T Easy vector  according to the manufacturer′s instructions. The recombinant plasmids were transformed into 66 and Unipro UGENE Version 1.14.067. For sequence analysis of Bdfru, the genomic scaffold (GenBank Acc. No. NW_011876307) was used to predict the gene structure. GenBank accession numbers of the references and the newly isolated sequences are in Supplementary Table Query DNA sequences were searched for homology using blast against the NCBI database for gene identification. Genomic DNA and cDNA sequences were aligned using the ClustalX2 programThe morphology of live embryos was observed during early development (0 to 9 h AEL) under differential interference contrast using an inverted confocal laser scanning microscope (Olympus FV1000). The collection of developing embryos, at each time point, was as previously described. The live embryos were gently dechorionated using 3% sodium hypochlorite solution for 5 min and then transferred in a drop of 1X phosphate buffer saline to a glass slide before observation and photography.This manuscript is a part of a research project which has been approved by Mahidol University-Institute Animal Care and Use Committee (no. MU-IACUC 2018/007). This research also complies with Biosafety Guildeline for Modern Biotechnology from Institutional Biosafety Committee under Pathogens and Animal Toxins Act, B.E. 2558 (2015); (Exempt2019-003).Supplementary Figures.Supplementary Tables.


N-isopropylacrylamide-co-vinyl-4,4-dimethylazlactone) attached to different permanently hydrophilic blocks, namely poly(ethylene glycol), poly, and poly, were successfully synthesized via reversible addition/fragmentation chain transfer radical polymerization (RAFT). Post-polymerization attachment of an amino-functionalized L-prolineamide using the azlactone ring-opening reaction afforded functionalized thermoresponsive block copolymers. Temperature-induced aggregation of the functionalized block copolymers was studied using dynamic light scattering. It was shown that the chemical structure of the permanently hydrophilic block significantly affected the size of the polymer self-assemblies. The functionalized block copolymers were subjected to an aldol reaction between p-nitrobenzaldehyde and cyclohexanone in water. Upon temperature-induced aggregation, an increase in conversion was observed. The enantioselectivity of the polymer-bound organocatalyst improved with an increasing hydrophilic/hydrophobic interface as a result of the different stability of the polymer aggregates.Micellar catalysts with a switchable core are attractive materials in organic synthesis. However, little is known about the role of the shell forming block on the performance of the catalyst. Thermoresponsive block copolymers based on poly( Increasing sustainability in chemical reactions, chemical processes, and materials is one of the major challenges in modern times. The design of energy-efficient chemical reactions using recyclable catalysts and avoiding toxic reaction media are key aspects of green chemistry . CatalysOne strategy for catalyst immobilization involves the attachment of the catalyst to the polymeric carrier. Such post-polymerization modification of a polymer usually requires the presence of reactive functional groups. Functional polymers based on isocyanides , epoxideN-isopropylacrylamide) (PNIPAAm) and poly (PDMAAm) were described by Levere et al. 0/[AIBN]0 = 50/1/0.1. The resulting reaction mixture was purged with nitrogen for 20 min, before the Schlenk tube was immersed into an oil bath at 70 °C. After 24 h, the polymerization was stopped by freezing the reaction mixture with liquid nitrogen. After thawing, 1,4-dioxane was removed under reduced pressure and the polymer was isolated by precipitation in n-hexane at room temperature. The poly (PSKA) was obtained as a sticky oil and used for the copolymerization as a macro-chain transfer agent.A Schlenk tube with a septum and magnetic stirrer was charged with SKA , DMP , AIBN , and solvent 1,4-dioxane (5.3 mL) in the ratio of [SKA]For the copolymerization, the previously obtained PSKA-based macro-chain transfer agent (0.35 g), NIPAAm , VDMA , and AIBN  were dissolved in 1,4-dioxane (5 mL). The reaction mixture was purged with nitrogen for 20 min, and the Schlenk tube was subsequently immersed into the oil bath. The polymerization proceeded at 70 °C for 24 h. Afterwards, the reaction was stopped by freezing the reaction mixture with liquid nitrogen. 1,4-Dioxane was removed under reduced pressure and the polymer was isolated by a series of precipitations in cold diethyl ether. The block copolymer was obtained as light-yellow powder after drying in vacuo.1H-NMR : δ = 1.13 ; 1.39 ; 1.57–2.49 ; 3.88 ; 3.88–4.18 ; 4.23–4.34 0/[DMP]0/[AIBN]0 equal to 50/1/0.1. The resulting reaction mixture was purged with nitrogen for 20 min. Subsequently, the Schlenk tube was immersed into an oil bath. The polymerization proceeded overnight at 70 °C. The reaction was stopped by freezing of the mixture with liquid nitrogen. After thawing, the yellow solid reaction mixture was dissolved in THF (2 mL) and purified by precipitations in cold diethyl ether. After drying under fine vacuum, PDMAAm was obtained as a light-yellow powder.DMAAm , DMP , and AIBN  were added to a Schlenk tube with a septum and magnetic stirrer, in the ratio of [DMAAm]n-hexane. The block copolymer was obtained as yellow powder after drying under fine vacuum.For the copolymerization, the PDMAAm-based macro-chain transfer agent (0.5 g), NIPAAm , VDMA , and AIBN  were dissolved in 1,4-dioxane (5 mL), and the resulting mixture was purged with nitrogen for 20 min. Polymerization was initiated by placing the Schlenk tube in the oil bath. The reaction proceeded overnight at 70 °C. The polymerization was stopped by freezing the reaction mixture with liquid nitrogen. 1,4-Dioxane was removed under reduced pressure and the polymer was purified by a series of precipitations in cold diethyl ether and 1H-NMR : δ = 1.14 ; 1.43–2.35 ; 2.90 ; 4.00  b-P(NIPAAm-co-VDMA) was synthesized following a previously described procedure [0/[VDMA]0/[PEG-CDPA]0 = 420/20/1. The reaction mixture was purged with nitrogen for 20 min and the Schlenk tube was subsequently immersed into an oil bath at 70 °C to start the copolymerization. After 24 h, the reaction was stopped by freezing the reaction mixture with liquid nitrogen. 1,4-Dioxane was removed under reduced pressure, and the polymer was purified by a series of precipitations in cold diethyl ether and finally dried under fine vacuum.PEG-rocedure . A Schle1H-NMR : δ = 0.98–1.23 ; 1.46–2.40 ; 3.63 ; 4.0 ® 6 Dialysis Membrane MWCO 1000, Spectrum Chemical, New Brunswick, NJ, USA). The modified polymers were obtained as white powder after freeze-drying.Modification of the azlactone-containing thermoresponsive block copolymers was carried out as previously described . In a 25b-P(NIPAAm-co-VDMA-prolinamide) block copolymer was dissolved in a mixture of 10 mL THF and 15 mL glacial acetic acid. The solution was heated to 90 °C and stirred for 6 h. During the reaction time, 24 mL deionized water was added portion-wise. The solvent was evaporated, residual acetic acid was removed by subsequent dialysis against 0.01 M sodium hydroxide solution and water , and the resulting polymer solution was freeze-dried.Deprotection of the solketal group was done according to a literature-known procedure . A mass p-NBA and 12 µL CH were added, and the mixture was stirred for 10 min prior to being filled into a cuvette. Dynamic light scattering analysis afforded the particle size as a function of temperature. The measurements were performed on a Zetasizer ZS from Malvern Instruments . The instrument was operated in backscattering mode. The temperature program covered a range of 20 °C to 65 °C in intervals of 1 K. The measurements were carried out 3 times at each temperature.A mass of 5 mg of the amphiphilic block copolymer was dissolved in 10 mL of distilled water. Being left for stirring overnight at room temperature, the resulting solution was subsequently filtered through a syringe filter . Then, 3 mg of p-NBA, 120 μL (1.16 mmol) CH, and a functionalized block copolymer carrying 16 µmol immobilized organocatalyst were suspended in deionized water. The resulting reaction mixture was stirred continuously for 10 min at room temperature and subsequently immersed into an oil bath at a certain temperature to start the catalytic reaction. After the reaction, the reaction mixture was diluted with water (10 mL) and extracted 3 times with diethyl ether. The organic phase was dried over MgSO4 and the solvent was removed under reduced pressure. The crude product mixture was analyzed using 1H-NMR spectroscopy and HPLC on a chiral stationary phase. The functionalized block copolymers dissolved in an aqueous phase can be isolated by freeze-drying.The amount of functionalized block copolymers used for the aldol reaction depends on the relative block copolymer composition and the molar mass of the organocatalyst (Table 3). In general, 30 mg (200 µmol) Three different block copolymers were synthesized by RAFT polymerization. For all three block copolymers, the thermoresponsive block comprised a copolymer of NIPAAm and VDMA. Meanwhile, the PNIPAAm provided thermoresponsivity, and the azlactone moiety was used for post-polymerization attachment of an amino-modified organocatalyst. The chemical nature of the hydrophilic block was varied from PDHPA and PDMAAm to PEG. Block copolymer synthesis started with the formation of the hydrophilic block . Accordi1H-NMR spectroscopy and SEC (Mn) of the block copolymers range between 39.7 and 55.9 kg/mol relative to a calibration based on PMMA standards. Although the molar mass dispersity seems to be high (Ð > 1.40), the molar mass distributions of the block copolymers are uniform . The slightly exothermic ring opening reaction will take place with primary amines and thiolates in the absence of any catalyst. In the present work, the post-polymerization modification of PVDMA was carried out using an amino-functionalized ineamide . AccordiOC 3 see . The rea1H-NMR spectroscopy, as previously reported [Finally, the solketal moiety was hydrolyzed using glacial acetic acid, thus yielding the hydrophilic PDHPA block . The sucreported .L-prolineamide organocatalyst and comprising three chemically different permanently hydrophilic blocks were available (1H-NMR spectroscopy (b-P(NIPAAm-co-VDMA-prolinamide) was calculated by comparing the characteristic signals corresponding to CH of PNIPAAm (at 3.84 ppm), the CH2-CH2 of PEG (at 3.51 ppm), and the CH2 of the proline-moiety (at 2.75 ppm). The molar copolymer composition of PDHPA-b-P(NIPAAm-co-VDMA-prolinamide) was calculated by comparing the characteristic signals corresponding to CH of PNIPAAm (at 3.84 ppm), the OH of PDHPA (at 4.59 and 4.78 ppm), and the CH2 of the proline-moiety (at 2.87 ppm). For the modified block copolymer PDMAA-b-P(NIPAAm-co-VDMA-prolinamide), the molar composition was determined by comparing the integral under the peaks attributed to the CH of PNIPAAm (at 3.84 ppm), the CH3 of PDMAA (at 2.79 ppm), and the CH of proline-moiety (at 3.51 ppm).At this step, three thermoresponsive block copolymers bearing the immobilized vailable . As the troscopy . The molThe molar content of the immobilized L-prolinamide organocatalyst was found to be in the range of 3–5 mol%. However, considering a targeted azlactone content of 5 mol%, the experimentally verified organocatalyst content of 3–5 mol% indicates that the nucleophilic ring-opening reaction of the azlactone by the organocatalyst did not proceed quantitatively. Approximately 60–95% of the available azlactone moieties were functionalized with the organocatalyst. Still, this degree of functionalization seems reasonable taking into account that the reaction was conducted in methanol. The application of the immobilized organocatalysts to an asymmetric aldol reaction in water requires knowledge of the catalyst loading, which is the number ratio of catalyst to substrate. Due to the dispersity in molar mass, polymer concentrations are usually mass-related. Therefore, the mass fraction of the organocatalyst was also calculated based on the molar block copolymer composition. Thus, the mole number of immobilized organocatalyst can be calculated from the applied polymer mass. According to the differences in the organocatalyst structure, the mass-related catalyst content differs between 9 and 12% w/w.p-nitrobenzaldehyde (p-NBA) and cyclohexanone (CH) was studied using dynamic light scattering (DLS). The overall ratio between functionalized block copolymer, p-NBA, and CH in this heterogeneous system matched the conditions for the later organocatalytic reactions. However, the overall concentrations had to be decreased in order to achieve evaluable scattering intensities in the DLS experiment.Thermoresponsive polymers undergo significant changes in their properties due to small changes in ambient temperature . TherebyDh,app, black dots) between sample POC 1 on the one hand and samples POC 2 and POC 3 on the other hand. For POC 1, at low temperatures, mainly small species with Dh,app ≈ 10 nm can be observed. Around the CPTT of 35 °C, a rapid increase in Dh,app shows the formation of larger aggregates with 600–1400 nm in size. The extensive scattering of Dh,app indicates the presence of differently sized species. By contrast, for samples POC 2 and POC 3, a formation of larger aggregates 250–600 nm in size at temperatures below CPTT can be seen. Around the CPTT, a significant decrease in Dh,app indicates the formation of smaller particles with less than 100 nm in size. The CPTT for samples POC 2 and POC 3 is around 32 °C. The broadness of particle size distributions characterized by the polydispersity index  is significantly reduced by exceeding the CPTT for all three samples. Thus, above CPTT, particles with narrower size distributions are formed. The smallest PDI value of 0.15 was observed for sample POC 3 at temperatures above 50 °C. p-NBA and CH are almost insoluble in water, thus leading to a heterogeneous system. However, the functionalized block copolymers POC 2 and POC 3 are capable of stabilizing larger substrate droplets even below CPTT. Therefore, an emulsion with larger droplets can be seen in the DLS experiment. Due to the amphiphilic character of the block copolymers above CPTT, the stabilization of the hydrophobic substrate domains becomes even better, resulting in the formation of micelles solubilizing the hydrophobic substrates. The PEG-based functionalized block copolymer POC 1 cannot stabilize the hydrophobic substrates below CPTT. Hence, mainly single dissolved block polymer chains with Dh,app ≈ 10 nm can be seen. Above the CPTT, the amphiphilic character improves the stabilizing properties of the block copolymer, leading to the formation of stabilized substrate domains. However, the dimension of these stabilized substrate domains is much larger compared to the micellar systems of POC 2 and POC 3.p-NBA and CH at different temperatures (dr) was determined by 1H-NMR spectroscopy and HPLC. The enantiomeric excess (ee) of the anti and syn adducts was determined by HPLC using a chiral stationary phase. The reactions were carried out at 25 °C and 40 °C, which are below and above the CPTT of the functionalized block copolymers, respectively. The molar ratio of immobilized organocatalyst to p-NBA was chosen to be 8 mol%. p-NBA conversions of 10% at maximum within 24 h. Above the CPTT, a significant increase in the p-NBA conversion was achieved. By reducing the volume of water and, thereby, increasing the concentration of substrates and functionalized block copolymers, the conversion could be optimized to 74% for POC 1 and 83% for POC 2 and POC 3. The diastereomeric ratio of the products did not show a significant dependency on temperature or concentration, whereas the ee values significantly improved by raising the temperature above the CPTT and increasing the concentration of the substrate and functionalized block copolymer. Gruttadauria et al. reviewed the role of water for organocatalytic reactions [ee values of POC 1 can be attributed to the larger aggregates formed above CPTT and the resulting low interface area.Finally, the functionalized block copolymers were subjected to an organocatalytic aldol reaction between eratures . This reeactions . Accordieactions . The lowp-NBA and CH in aqueous media. Dynamic light scattering revealed that temperature-induced self-assembly of the functionalized block copolymers lead to the formation of nanoreactors with the hydrophobic substrates in the core and the immobilized organocatalyst at the hydrophilic/hydrophobic interface. However, the dimension of the formed polymer aggregates significantly depended on the chemical structure of the permanently hydrophilic blocks. Whereas the presence of PEG as a stabilizing block afforded larger aggregates , the use of PDMAAm and PDHPA as a hydrophilic block led to the formation of micellar aggregates . The temperature-induced self-assembly of the block copolymers significantly enhanced the reaction rates of the aldol reaction. Diastereoselectivity of the immobilized catalyst was not affected by polymer self-assembly, whereas enantioselectivity clearly improved upon the formation of the nanoreactors. The size of the polymer aggregates also affected the ee values. Within the micellar aggregates, higher ee values were observed compared to the larger aggregates. This effect can be attributed to the larger hydrophilic/hydrophobic interface formed in the micellar system, thus improving stereoselectivity of the organocatalyst.Thermoresponsive block copolymers based on PNIPAAm attached to different permanently hydrophilic blocks, namely PEG, PDMAAm, and PDHPA, were successfully synthesized via RAFT polymerization. Additionally, the thermoresponsive block contained azlactone moieties used for post-polymerization attachment of an amino-functionalized organocatalyst based on L-prolinamide. These functionalized thermoresponsive block copolymers were subjected to the aldol reaction between


ALL resulted in a complete remission (CR) rate of 81% and an overall median survival of 7.7 months with reduced toxicity. Similarly, blinatumomab, the first FDA-approved bispecific antibody (BsAb), produced a 44% complete response rate and an overall median survival of 7.7 months in a widely treated ALL population. In addition, approximately 80% of patients getting complete remission with evidence of minimal residual disease (MRD) achieved a complete MRD response with the use of blinatumomab. These results highlight the great promise of antibody-based therapy for ALL. How to reasonably determine the place of antibody drugs in the treatment of ALL remains a major problem to be solved for ongoing and future researches. Meanwhile the combination of antibody-based therapy with traditional standard of care (SOC) chemotherapy, chimeric antigen receptor (CAR) T-cell therapy and HSCT is also a challenge. Here, we will review some important milestones of antibody-based therapies, including combinational strategies, and antibodies under clinical development for ALL.Acute lymphoblastic leukemia (ALL) is a hematologic malignancy arising from precursors of the lymphoid lineage. Conventional cytotoxic chemotherapies have resulted in high cure rates of up to 90% in pediatric ALL, but the outcomes for adult patients remain suboptimal with 5-year survival rates of only 30%-40%. Current immunotherapies exploit the performance of antibodies through several different mechanisms, including naked antibodies, antibodies linked to cytotoxic agents, and T-cell re-directing antibodies. Compared with chemotherapy, the application of an antibody–drug conjugates (ADC) called inotuzumab ozogamicin in relapsed or refractory (R/R) CD22 The application of classical multi-agent chemotherapy in patients with ALL results in CR in more than 80% of patients. About 50% of newly diagnosed patients can achieve long-term disease control with further intensification or maintenance therapy. However 10% have initial refractory disease . What’s Paul Ehrlich, a German physician and scientist proposed the conception of antibodies as a “magic bullet” for selective targeting of malignant cells. Nevertheless, it took about a century to achieve the full potential of antibody therapy. Up to now, antibody-based therapies targeting leukemic cell surface antigens are major breakthroughs in the treatment of patients with ALL, changing the traditional treatment paradigms [Over 100 years ago, aradigms .Based on the excellent outcomes in patients with R/R B-cell ALL, this magic bullet has actually been incorporated into the frontline. Antibodies against some tumor-associated antigens (TAAs) have performed well in clinical trials and have successfully come to fruition, such as inotuzumab ozogamicin , 8 and BMonoclonal antibodies can be classified into three main groups according to their construction: naked antibodies, ADCs, and T-cell re-directing antibodies. These agents bind to known surface cell antigens present on the ALL blasts and mediate cell death through a variety of mechanisms that are specific to their target antigens and construct. Naked antibodies bind directly to the surface cell antigen and mediate cell lysis through antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and induction of apoptosis. A variety of ADCs has also been developed that link a monoclonal antibody to a potent cytotoxin or radioisotope, knows as payloads. These conjugated antibodies are internalized upon binding to the surface cell marker, leading to cell death through the release of the toxic payload. Bispecific antibodies (BsAb) have attracted significant attention in antitumor immunotherapy . Based o+ precursor B-ALL is considered standard of care. In phase III multi-center randomized GRAALL-2005/R [p = 0.04). The 2-year OS was 71% in the rituximab group versus 64% in the control group, although not statistically significant (p = 0.10). Sensitivity analyses with monitoring during HSCT showed a statistically significant improvement in OS in the rituximab group . This study demonstrated that addition of rituximab to conventional chemotherapy improved the patient outcome with B-cell ALL in terms of EFS for a certainty. Ofatumumab is a second-generation anti-CD20 monoclonal antibody, that leads to more effective ADCC and CDC than rituximab, which is associated with its binding to a proximal small loop on the CD20 antigen [Monoclonal antibodies for many established cell surfaces which are highly expressed on ALL blasts had achieved precise results. The CD20 antigen can be found in about 30–50% of B-cell ALL, while CD19 and CD22 are present on the cell surface in over 90% of B-cell ALL , 13. RitL-2005/R  trial, p antigen , 19. In Given the fact that nearly 90% of B lymphoblasts in ALL express CD22, which rapidly internalized upon ligand binding , researc+ refractory B-ALL [et.al recently described [The latest analyses show that CD38 is a promising therapeutic antigen for acute leukemia . Daratumry B-ALL , and anory B-ALL . The appescribed  the clinescribed ). The paescribed .CD22 is a 135 kDa sialoglycoprotein that is generally considered as an important B-lineage surface antigen. There are further studies conducted to better understand the immunobiology and metabolism of CD22 to aid in the development of CD22-directed therapies for the treatment of B-lymphoid malignancies. In a flow-cytometric cell surface expression study of 104 ALL cases, there was a significant positive correlation between CD22 expression and ALL at 96% (considering the expression of  > 20% in blast cells as positive) . CD22 unIO binds to CD22 and is internalized to release calicheamicin, a cytotoxic payload that binds to double-stranded DNA. Upon antigen binding, the ALL cell endocytoses IO and the acidic environment of the lysosome dissolves the linker protein, thus releasing the calicheamicin toxin intracellularly. In vitro studies have shown that cells required CD22 expression for the uptake of IO, but continuous saturation of the receptor was not a necessity for apoptosis, suggesting that multiple low IO dosages may be effective .2 per cycle in a fractionated weekly dosing (0.8 mg/m2 on day 1 and 0.5 mg/m2 on days 8 and 15 per cycle). The chemotherapy regimens were either the FLAG regimen, a high-dose cytarabine-based regimen, or cytarabine plus mitoxantrone.IO was subsequently compared with standard salvage in the INO-VATE study, a phase III study of 326 patients with R/R B-ALL . All patp < 0.001) and an MRD negativity rate of 78 versus 28% (p < 0.001) by flow cytometry. Compared with the SOC group, more patients who received IO underwent HSCT . The median PFS for IO and for SOC was 5 versus 1.8 months (p < 0.001), and the median OS was 7.7 versus 6.7 months (p = 0.04), respectively. Significantly higher remission rates were observed in all patients regardless of bone marrow blast percentage, CD22 expression, prior HSCT, or karyotype, except for Ph-positive ALL patients who did not preferentially benefit from the 2 therapies. Hepatotoxicity was the most common event in patients treated with IO. Veno-occlusive disease (VOD) was reported in 15 patients (11%) in the IO group. Most cases 10–15 patients occurred after HSCT and the median time of development was 16 days (3–39). In August 2017, based on the data of the INO-VATE study, the US FDA approved IO (BESPONSA) for the treatment of adults with R/R B-cell precursor ALL (BCP-ALL).The CR/CR with incomplete hematologic recovery (CRi) and MRD negativity rate was significantly higher in the IO arm with CR/ CRi rates of 81 versus 29%  is an ADC comprising of a humanized anti-CD19 antibody, stochastically conjugated through a cathepsin-cleavable valine-alanine linker to SG3199, a pyrrolobenzodiazepine (PBD) dimer-containing toxin. The mechanism of SG3199 for DNA crosslinking contributes to persistence in cells , and SG3 studies . In precl models . A phasel models , demonstHuman CD25, the α-chain of the heterotrimeric interleukin-2 receptor, is a critical component in regulating the immune system –42, and CD123, the alpha chain of the interleukin-3 (IL-3) receptor, is the major low-affinity subunit of the IL-3 receptor and promotes high-affinity binding to IL-3 when co-expressed with the beta subunit. IL-3 is mainly produced by T lymphocytes, and it regulates the production of hematopoietic cells by stimulating cell cycle progression, differentiation, and inhibiting apoptosis. Early studies have shown that IL-3 plays a key role in the development of leukemia by allowing leukemia cells to escape programmed cell death and grow autonomously . The pot+ T effector memory (TEM).BiTE is a relatively mature kind of BsAb obtained by ligating anti-CD3 single-chain Fv (scFv) with its counterpart of various anti-tumor cell surface antigens through peptides, which can simultaneously bind T cells to tumor cells and induce continuous attack of the target without T-cell apoptosis or anergy. Blinatumomab is the first FDA and EMA approved BiTE for the treatment of R/R ALL. It is a small (55 kDa) single-chain peptide that links two antibody variable regions directed against CD3 and CD19. Cytolytic synapsis formed, T cells can be activated without costimulatory molecules. Blinatumomab results in the proliferation of CD8-positive T cells with a predominance of cytotoxic CD8p < 0.001), greater MRD negativity (76 vs. 48%) and longer median OS  in comparison to SOC chemotherapy [− ALL [+ ALL [Blinatumomab has shown encouraging results in phase I/II clinical trials in R/R B-cell ALL, especially in the setting of low tumor burden , 50. Theotherapy . This bey [− ALL  or Ph+ AL [+ ALL , the two–3. The primary endpoint was MRD negativity after one cycle. A total of 91 of the 113 evaluable patients obtained a complete MRD response of 78% (82 patients were attained after the first cycle). The medians of RFS and OS for the group were 18.9 and 36.5 months, respectively. The tolerability of blinatumomab was similar to that reported in previous trials. Based on these results, the regulatory authorities in the US extended the authorization of blinatumomab to MRD-positive BCP . Similar studies using blinatumomab in other settings are also in the pipeline; for example, post-transplant maintenance to prevent relapse after HSCT (NCT02807883), which has a parallel in the use of post-transplant TKI (tyrosine kinase inhibitor) therapy in Ph+ B-ALL.To date, several mechanisms have been addressed to explain resistance or relapse after blinatumomab strategies. These factors comprise the presence of extramedullary disease , higher IGM-2323 is a CD20/CD3 bispecific IgM antibody designed to treat patients with B cell Non-Hodgkin’s lymphoma (NHL) and other B cell malignancies. Its potential antineoplastic activity manifests itself. Instead of binding to one or two TAA molecules on the surface of the cancer cell, IGM-2323 has 10 binding units to CD20 and one binding unit to CD3. Therefore, it can bind to CD20-expressing cancer cells with higher avidity in comparison with the IgG type. This may include those clinical circumstances in which CD20 expression has been reduced due to prior treatment with other anti-CD20 antibodies, such as rituximab. Two mechanisms are employed aimed at killing cancer cells. One is T cell-directed cellular cytotoxicity (TDCC) and the other is CDC. Compared to IgG format BiTEs, IGM2323 appears to induce less cytokine release, and less CRS, associated with TDCC. Currently, there is only one study reporting the safety and pharmacokinetics of IGM-2323 in subjects with R/R NHL. Therefore, its potential to cure more hematological malignancies, like acute leukemia, could be anticipated.Due to the small size of blinatumomab, it can reach the T cells and target membranes quickly, which also leads to its rapid clearance from the circulation , 57. The. [DART consists of two Fv fragments that form two unique antigen binding sites when they heterodimerize. Fv1 consists of VH from antibody A and VL from antibody B, whereas Fv2 is formed of VH from antibody B and VL from antibody A. Unlike BiTEs antibodies which are linked via a polypeptide linker, this combination allows DART to mimic natural interactions within IgG molecules. Compared to BiTE, DART molecules can also maintain potency when administered both in vitro and in vivo , 62. Moo.  comparedCompared to natural antibodies, the renal clearance of small-sized scFv lacking the Fc region is significantly higher. Tandem diabodies (TandAbs) have emerged to address the size problem. These tetravalent bispecific antibody provides two binding sites for each antigen to maintain the avidity of each natural bivalent antibody , 64. Fur. [With the evolution of antibody technology, CD28 co-stimulation provides a novel choice for therapeutic interventions. Recently, Wu et al.  publisheTo confirm the enhanced activity from costimulatory domain of trispecific antibodies, the group made versions of different combinations of all three binding domains that were mutated and tested them in "humanized" model mice with human T cells and human myeloma cells. Functional targeting of the CD28 domain enhanced T cell activation above that showed using antibodies lacking this domain. The results found that both proliferation of T cells and expression of the anti-apoptotic protein Bcl-xL were significantly enhanced, supporting the authors' hypothesis, that is, having costimulatory signals would prevent T cell apoptosis, even at the lowest antibody dose tested. The method of introducing the costimulatory domain has also been applied in chimeric antigen receptor T-cell (CAR-T) therapy at the same time. The main limitation of this study is the cytokine release syndrome (CRS) resulting from highly stimulated immune system.Currently, several novel antibodies can provide effective salvage therapy for patients with R/R ALL, with higher response rates and OS rates compared with conventional chemotherapy. Early addition of these new agents to the frontline can be considered promising to further consolidate the therapeutic efficacy of relevant patients, while reducing the number of cytotoxic chemotherapies required to achieve a durable response. Such regimens can improve tolerability and reduce treatment-related morbidity and mortality. In R/R cases, correlative combination studies are ongoing to improve the results of the single-agent application of Blinatumomab and IO. Some trials have corroborated this, especially for patients in first salvage . Althoug2 on days 1, 8 and 15, given in 4-weekly cycles. 14 patients, with a median age of 62 years, were treated in the study. Overall, 79% of patients achieved CR/CRi, and 91% of responders achieved a complete cytogenetic response, 73% achieved MRD negativity. No BCR-ABL was detected in 55% (6/11) of responders. The median OS and EFS were 8.2 and 8.1 months, respectively.Blinatumomab was demonstrated in the phase II ALCANTARA study to be safe and effective in patients with Ph-positive ALL . In comb+ ALL patients of all ages. The general story is that, 5 relapses have been observed , the 12-month OS and DFS are 94.2% and 87.8%. A significantly inferior DFS  was observed in IKZF1plus cases. So deep molecular response improved throughout therapy, but patients carrying IKZF1 plus remains a clinical challenge.The GIMEMA group has recently updated their results from D-ALBA , the firA recent exciting result via chemo-free induction and consolidation first-line strategy with dasatinib and blinatumomab  brings oNovel antibody-based drugs such as blinatumomab and IO are some of the most exciting and promising agents approved for patients with ALL. However, the present study has several limitations that should be noted. Firstly, the short duration of response and survival outcomes, and the efficacy which is mainly dependent on the percentage and density of the antigen expression, as well as the persistence of antigen expression after repeated immunotherapy exposure. This suggests that antibody-based therapies alone cannot treat ALL. Therefore, we still need HSCT, traditional chemotherapy, TKIs, and even CAR-T therapy to achieve comprehensive treatment of ALL.To know that patients are likely to quickly progress with each therapy, we need to consider which therapy to use first or how to sequence or combine the therapies. Therefore, clinical trials of low-intensity comparing IO with or without blinatumomab in first-line and R/R Ph-negative elderly patients are encouraging –77. RetrBesides, with the latest developments and approval of CAR-T cell therapy for ALL in recent years, how to rationally use the two new drugs has also attracted significant attention. Up to now, it is still not clear how prior blinatumomab therapy affects the ongoing anti-CD19 CAR-T therapy. There are also concerns that blinatumomab causes the loss of CD19 antigen and disrupted CD19 membrane export. However, remission of such a sequential application is still possible , 80, 81.With the development of antibody-based therapies such as monoclonal antibodies, ADCs, bispecific, tri-specific, and even multi-specific antibodies, treatment options are widely expanded. As discussed above, antibody-based therapies still face challenges in determining the best-optimized treatment combination. However, successful clinical studies reporting combinations with other immunotherapies such as CAR-T therapy, and T-ALL with the few available drugs, is a reason to be optimistic that antibody-based treatment approaches may eventually become a success story in ALL therapy.


The mechanical head-withdrawal threshold (MHWT) was significantly reduced following inferior alveolar nerve transection (IANX) in rats. Nitrate and nitrite synthesis was dramatically increased in the trigeminal ganglion (TG) at 6 h after the IANX. The relative number of neuronal nitric oxide synthase (nNOS)-immunoreactive (IR) cells was significantly higher in IANX rats compared to sham-operated and N-propyl-L-arginine (NPLA)-treated IANX rats. On day 3 after NPLA administration, the MHWT recovered considerably in IANX rats. Following L-arginine injection into the TG, the MHWT was significantly reduced within 15 min, and the mean number of TG cells encircled by glial fibrillary acidic protein (GFAP)-IR cells was substantially higher. The relative number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased in IANX rats. In contrast, after NPLA injection into the TG, the relative number of GFAP-IR cells was considerably reduced in IANX rats. Fluorocitrate administration into the TG significantly reduced the number of GFAP-IR cells and prevented the MHWT reduction in IANX rats. The present findings suggest that following IANX, satellite glial cells are activated via nitric oxide (NO) signaling from TG neurons. The spreading satellite glial cell activation within the TG results in mechanical hypersensitivity of face regions not directly associated with the trigeminal nerve injury. It is well known that ectopic orofacial pain occurs after trigeminal nerve injuries such as tooth extraction, tooth pulpectomy, maxillofacial bone fractures, or orofacial cancers . Injury-12 receptor, suggesting that ATP is released from TG neurons and binds to the P2Y12 receptor to activate satellite glial cells. SP, calcitonin gene-related peptide (CGRP), and tumor necrosis factor α are also released from TG neurons and involved in the activation of satellite glial cells [Recently, it has been reported that satellite glial cells are activated in the TG following trigeminal nerve injury . Satellial cells . FurtherNitric oxide (NO) is known to be synthesized in neurons from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). At room temperature, NO exists as a free radical gas, passes freely through biological membranes, and acts as a cell signaling factor . NeuronaBased on these findings, we hypothesized that the functional interaction between neuronal ganglion and satellite glial cells via NO-dependent mechanisms plays a pivotal role in persistent ectopic pain in the orofacial region associated with trigeminal nerve injury. To evaluate the mechanisms underlying ectopic orofacial pain after trigeminal nerve injury, we examined changes in mechanical sensitivity in the whisker pad skin of an IANX rat model. Moreover, SGC activation and nNOS expression in TG neurons and their localization within the TG were examined immunochemically. To determine whether NO-dependent SGC activation is involved in ectopic mechanical allodynia in the orofacial region following IANX, selective SGC or nNOS inhibitors were applied to the TG. Additionally, L-arginine was administrated to the TG to test whether L-arginine-induced activation of SGCs is involved in the ectopic orofacial pain. Mechanical stimulation was applied to the whisker pad skin, ipsi- or contralateral to the site of the IANX or sham surgery . The mecTo quantify NO levels in the TG, we conducted nitrate and nitrite measurements in the TG. We observed significantly increased amounts of nitrate and nitrite in the TG of IANX rats compared to those of sham rats at 6 h after the operation . These lNext, we studied the expression of nNOS-IR in TG cells after IANX . We obseL-arginine is a substrate for the enzyme nNOS that synthetizes NO . Thus, wWe observed many GFAP-IR cells encircling TG cells 6 h after L-arginine but not vehicle administration into the TG a. The meWe also examined nNOS-IR cells surrounded by GFAP-IR cells in the TG of IANX and sham-operated rats. Many nNOS-IR cells were encircled by GFAP-IR cells in the TG of IANX rats, but only a few of these cells were observed in sham-operated rats a–f. The In sham-operated rats, only a small number of GFAP-IR cells were observed in the TG a. By conFinally, we studied the expression of GFAP in the TG of IANX rats in the presence and absence of FC. A small number of GFAP-IR cells were observed in the TG of sham-operated rats, and no TG cells were encircled by GFAP-IR cells a–c. In contrast, many GFAP-IR cells were observed in vehicle-treated IANX rats, and many of them encircled 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled TG cells d–f. The The findings of the present study are summarized as follows. (1) The head-withdrawal threshold to mechanical stimulation of the whisker pad skin was significantly reduced following IANX. (2) Nitrate and nitrite levels were significantly increased in the TG after IANX, and nNOS was also considerably expressed in these neurons. (3) Inhibition of nNOS expression caused suppression of glial cell activation and reduction in MHWT in IANX rats. (4) Following L-arginine injection into the TG, the MHWT was considerably reduced, and the mean number of TG cells encircled by GFAP-IR cells was significantly higher in L-arginine-treated rats. (5) The number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased following IANX. (6) The blockade of satellite glial cell activation caused significant recovery of the MHWT in IANX rats.These observations suggest that NO is a key molecule involved in neuron-satellite glial cell communication and associated with mechanical allodynia in the broad orofacial area following trigeminal nerve injury.Trigeminal nerve injury is known to cause mechanical allodynia and thermal hyperalgesia in the orofacial region, namely orofacial neuropathic pain . To evalWe observed a significant reduction in the MHWT following IANX, as reported in a previous study , indicatNO is thought to act as a neurotransmitter in peripheral and central nervous systems and is synthesized from L-arginine and oxygen by the enzyme NOS in ganglion neurons following nerve injury or tissue inflammation . NO is qNO is also known to enhance the release of SP and CGRP from C-fiber terminals, thus contributing to secondary hyperalgesia in tissues . FurtherAfter IANX, high-frequency discharges are generated in uninjured, as well as injured, neurons . It is aIn our study, nNOS positivity was detected in injured but not uninjured TG neurons of IANX rats. Furthermore, injection of the NO synthesis inhibitor NPLA into the TG caused a significant decrease in the number of nNOS-IR and GFAP-IR cells in the TG. Similarly, IANX-induced MHWT changes were significantly reversed following NPLA administration. This series of events underlying NO-mediated neuron-satellite glial cell communication causing the spreading of satellite glial cell activation into uninjured TG branch regions may be involved in the observed hyperexcitability of uninjured TG neurons following trigeminal nerve injury. In cell culture studies, CGRP secreted from neurons has also been reported to induce an increase in inducible NOS (iNOS) in SGCs via mitogen-activated protein kinase signaling pathways ,30. NO rn = 112, 7 weeks old, weighing 200–250 g) were used in this study. The animals were housed in cages (42 × 26 × 22 cm) that were kept at constant room temperature (23 ± 2 °C) and relative humidly (55 ± 5%) under conditions of a 12-h light/dark cycle (lights on at 7:00 a.m.) with free access to food and drinking water. The care and treatment of the animals used in this study were conducted according to the guidelines of the International Association for the Study of Pain [Male Sprague Dawley rats   administration of a saline solution containing 2.5 mg/kg butorphanol , 0.375 mg/kg medetomidine , and 2.0 mg/kg midazolam . The animals were placed on a warm mat (37 °C). The left facial skin and masseter muscle were incised, and the surface of the alveolar bone was exposed. The alveolar bone covering the inferior alveolar nerve was removed to expose the IANX, and was tightly ligated with a 4-0 silk thread at two points and transected between the two ligatures and repositioned in the mandible canal without a discernable gap between the cut nerve ends. For control, a sham operation was identical to that described above but without the transection. The incised skin was sutured using a 6-0 silk thread.Prior to behavioral testing, the rats were trained to keep their head protruding from a hole in a plastic cage while the left whisker pad skin was mechanically stimulated with von Frey filaments . To quantitatively evaluate the MHWT, we applied 4, 6, 8, 10, 15, 26, and 60 g of mechanical stimuli to the whisker pad skin. The MHWT was determined as the minimum stimulation intensity that evoked a head-withdrawal behavior in response to more than 3 out of 5 stimuli. The MHWT was measured for 5 d after IANX or sham operation. All behavioral tests were conducted under blinded conditions NO is known to exist in vivo as an unstable free radical gas that passes freely through biological membranes and acts as a cell signaling factor. Therefore, we measured serum NO metabolites (nitrate/nitrite) as a quantification of the NO level . The ratRats were anesthetized with sodium pentobarbital  and placed in a stereotaxic frame, the skull was exposed, and a small hole (diameter: 1 mm) was drilled directly above the location of the bifurcation between the 1st and 2nd branches of the TG (V1/V2) and the 3rd branch of the trigeminal nerve region (V3) of the TG. Through this hole, a guide cannula was implanted in the TG ipsilateral to the IANX or sham operation and fixed to the skull with stainless screws and dental acrylic cement. The coordinates for the target site of the TG were 9 mm below the skull surface, 2.8 mm anterior from the posterior fontanelle, and 2.7 mm lateral to the surgical suture. The rats were allowed to recover from the operation for a week. Fluorocitrate , an aconitase inhibitor that is taken up by glial cells and leads to inhibition of the glial tricarboxylic acid cycle, was dissolved in PBS (vehicle). For continuous FC or vehicle administration into the TG, rats were anesthetized, and a 31-gauge injection needle was inserted through the guide cannula 9.5 mm below the skull and was connected to an osmotic pump  filled with FC or vehicle by a soft micro-silicone tube. The osmotic pump was embedded in the dorsal body part of the rat. FC (1 µL/h) was administered for 3 d continuously from the osmotic pump.For vehicle  or N-propyl-L-arginine hydrochloride  administration into the TG, a 31-gauge injection needle was inserted in the TG 9.5 mm below the skull surface through the guide cannula under light anesthesia with 2% isoflurane in oxygen. Then, 5 µL of NPLA or vehicle was administered into the TG once per day for 3 d after IANX or sham operation. Saline as a vehicle or L-arginine  dissolved in saline was also administrated into the TG in naïve rats.To identify the TG neurons innervating the left whisker pad skin, 100 mg/mL 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine methane sulfonate  was used as a retrograde labeling tracer. DiI was injected into the skin of the left whisker pad with a 30-gauge needle 7 d before and 3 d after IANX or sham operation, rats with continuous administration of FC or vehicle, or rats with once per day for 3 d administration of NPLA or vehicle into the TG, were anesthetized with sodium pentobarbital (50 mg/kg. i.p.) and transcardially perfused with 100 mL saline followed by a fixative containing 4% paraformaldehyde in 0.1 M PBS at 4 °C (pH 7.4). The TGs were removed after perfusion, immersed in the same fixative, and transferred to 20% sucrose in 0.01 M PBS for 12 h at 4 °C for cryoprotection. The TGs were embedded in Tissue Tek  and stored at −20 °C for cryosection, and were cut on the horizontal plane along the long axis at a thickness of 10 µm. Every 10th section was selected, thaw-mounted on MAS-GP micro slide glass , and then dried at room temperature. After rinsing with 0.01 M PBS, the sections were incubated with mouse anti-GFAP monoclonal antibody  in 0.01 M PBS containing 4% normal goat serum and 0.3% Triton X-100 (Sigma-Aldrich) for 3 d at 4 °C or with goat anti-nNOS polyclonal antibody  in 0.01 M PBS containing 4% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C. After rinsing with 0.01 M PBS, the sections were reacted with AlexaFluor 488-conjugated goat anti-mouse IgG (1:200 in 0.01 M PBS) or AlexaFluor 594-conjugated donkey anti-goat IgG (1:200 in 0.01 M PBS) for 2 h at room temperature, respectively. After another rinse with 0.01 M PBS, sections were coverslipped with mounting medium  and examined using a fluorescence microscope BZ-9000 system . The number of DiI-labeled TG neurons encircled by GFAP-immunoreactive cells over 1/2 of the TG cell body was counted as GFAP-IR, and the number of nNOS-immunoreactive TG neurons expressing twice or greater the luminescence intensity relative to the background was counted as nNOS-IR. We also studied double immunohistochemistry with nNOS-IR and GFAP-IR cells in the TG. nNOS-IR cells encircled by GFAP-IR cells were counted in the TG of sham-operated and IANX rats, and the relative number of nNOS-IR cells encircled by GFAP-IR cells was calculated . No specific labeling was observed in the absence of primary antibodies.t-test for the quantitative analysis of nitrate and nitrite. The one-way repeated-measures analysis of variance (ANOVA) was followed by Bonferroni’s, Tukey’s, or Dunnet’s multiple comparison tests, as appropriate. Data were considered significant at p < 0.05.Behavioral data are presented as box and whisker plots. The box bottom and top indicate the lower and upper quartiles, respectively, and the lower and upper whiskers represent the minimum and maximum values, respectively. Other results are expressed as the mean ± SEM, and statistical analyses were performed using Student’s The present findings suggest that IANX leads to satellite glial cell activation via NO, which is released from TG neurons. The activation of satellite glial cells spreads to large areas within the TG and can affect uninjured branches of the trigeminal nerve, resulting in mechanical hypersensitivity of orofacial regions not directly associated with the trigeminal nerve injury. The present findings suggest that NO signaling in the trigeminal ganglion is a therapeutic target for the treatment of orofacial neuropathic pain.


Childhood obesity has become a global epidemic and the etiology of maladaptive ingestive behavior in children warrants further research. Mounting evidence suggests that the caudate is associated with body weight gain and obesity in adults. In adolescents, however, how caudate-related neural networks are associated with body weight gain is unclear because their central nervous systems are still developing.The current longitudinal resting-state functional magnetic resonance imaging (rs-fMRI) study was conducted to investigate the hypothesis that caudate-related neural networks have a role in weight gain in adolescents.The study included 20 healthy adolescents with a mean age of 17.5 ± 2.0 years and a mean body mass index of 20.6 ± 2.1 who underwent baseline rs-fMRI then follow-up rs-fMRI approximately 1 year later. Body mass index (BMI) was measured at both timepoints. Seed-based functional connectivity analysis was utilized to analyze caudate-related functional connectivity (FC) using the caudate as a seed. Associations between caudate-related FC and BMI at baseline were assessed, as were associations between change in BMI and caudate-related FC between baseline and follow-up.p < 0.05). Compared to the baseline, increased FC in the caudate-lateral prefrontal cortex at follow up were negatively associated with increased BMI (p < 0.05).At baseline, greater caudate-lateral prefrontal cortex FC was correlated with lower BMI (family wise error-corrected Given that the lateral prefrontal cortex and caudate are associated with inhibitory control, the caudate-lateral prefrontal cortex FC may have a preventive effect on weight gain in adolescents. The results of the current study suggest that developing inhibitory control would lead to the prevention of childhood obesity. Childhood obesity has become a global epidemic in the last three decades , and it Weight change is reportedly associated with structural and functional alterations in reward regions of the brain such as the midbrain, pallidum, orbitofrontal cortex , and espThe caudate is deep inside the brain near the thalamus, and is connected to parts of the brain associated with nearly all of its facets, including cognition, emotion, perception, and action-related structures and networks . The cauIt has been repeatedly reported that impulsivity is associated with binge eating that could be a cause of excessive weight gain  in youngThe study included 20 adolescents  with a mean age of 17.5 years  and a mean BMI of 20.6  at baseline who were recruited from the metropolitan area of Tokyo. All participants and parents/guardians of the high school or middle school participants provided written informed consent, and the study was approved by the Ethics Committee of the Graduate School of Arts and Sciences at the University of Tokyo . All participants were healthy and had no history of psychological or neurological disorders or head injury. Detailed demographic data are shown in 2). We also collected self-reported weight and height on the scanning day. We calculate BMI using self-reported weight and height. There was no significant difference between self-reported BMI and measured BMI . Therefore, we assumed that the participants’ BMI did not significantly change within a month from the rs-fMRI scanning day.All participants underwent rs-fMRI at baseline and approximately 1 year or more thereafter . Within 1 month before or after each rs-fMRI session, each participant visited the laboratory and their weight and height were measured by trained experimenters (YN or SO) using a multi-frequency body composition meter  to calculate BMI  , which wApproximately three to 4 h after a meal, the level of appetite generally returns to almost the same level as that of the pre-meal appetite . Since cThe participant was instructed to relax and lie still in the scanner while remaining calm and awake. During rs-fMRI scanning, the participant was instructed to fix their gaze on a white cross on the black screen, which was projected on a mirror attached to the head-coil from the monitor that was set behind the bore of the MRI machine.∗-weighted images reflecting blood oxygen level-dependent signals were acquired using gradient-echo echo-planar imaging  in ascending order (10 min and 10 s). Anatomical images were acquired using a T1-weighted 3D MPRAGE protocol .Images were acquired on a 3 Tesla MRI scanner  using a 64-channel head/neck coil. T2Resting-state functional magnetic resonance imaging data were preprocessed using FSL (version 6.0)  and AFNIBased on a previous report , 3-mm spt-test.We tested for gender differences in BMI at baseline and follow-up, and in the change in BMI, using a two-sample t-tests were conducted on individual connectivity maps derived from baseline and follow-up data to compare caudate-related FC.First, paired z > 3.1. Clusters were then formed, associated p values were calculated, and p values that were > 0.05 (family wise error (FWE) corrected) were disregarded.Then, to assess the correlation between BMI and individual connectivity maps at baseline and follow-up, a group-level voxel-wise linear regression analysis with BMI as a covariate of interest was performed on individual caudate-related connectivity maps. To control for variance in age, age was included as a covariate of no-interest in the regression analysis. The cluster-forming threshold was set at z-values) were extracted from connectivity maps at baseline and follow-up and compared. Further, correlation coefficients between extracted FC values and BMI were compared at baseline and follow-up using the “cocor” package version 1.1-3 in R , 95% confidence intervals (CI) were calculated based on 1000 samples with the percentile bootstrap method implemented in the toolbox.Changes in extracted FC values between baseline and follow-up (follow-up > baseline) were calculated, and the correlations between changes in extracted FC values and change in BMI (follow-up > baseline) were calculated using the Robust Correlation Toolbox in MATLAB R2020a  . Robust p = 0.74, t = −0.34). The change in BMI was not significantly associated with the change in age .The mean change in BMI was 0.25 ± 2.53 (range −4.33 to 6.53), and there was no significant difference in the change in BMI between male and female participants  or at follow-up .There was no significant gender difference in BMI at baseline . There were no significant differences between ratings for hunger and fullness at baseline  or follow-up , suggesting that participants were neither hungry nor full at either of their rs-fMRI sessions.The mean hunger rating at baseline was 4.6 ± 1.2, and at the follow-up timepoint, it was 4.1 ± 1.3. The mean fullness rating at baseline was 4.2 ± 1.2, and at the follow-up timepoint, it was 4.8 ± 1.2. Ratings for hunger and fullness at baseline did not differ significantly from the corresponding ratings at follow-up    = , z = 4.09, PFWE–corrected = 0.0030, cluster size = 165 voxels].Body mass index was inversely correlated with caudate-related FC in the inferior frontal gyrus (IFG), the opercular part  . After cThere was no significant positive nor negative correlation between caudate-related FC and BMI at follow-up.The linear relationship between caudate-related connectivity maps and BMI did not differ between baseline and follow-up.p = 0.61, t = −0.94 for FC in the IFG and p = 0.34, t = −0.97 for FC in the SFG).Extracted caudate-related FCs in the IFG or SFG at baseline did not differ from those at follow-up .Correlations between extracted FCs in the IFG or SFG and BMI at baseline were greater than those at follow-up  or SFG , but change in BMI .Time of intervals between baseline and follow-up rs-fMRI sessions were not significantly correlated with caudate-related FC in IFG  from the IFG  = 3) and from the SFG   . These rp = 0.003, r = 0.63 for attentional subscale; p = 0.033, r = 0.48 for motor subscale) and increased attentional subscale with age at follow-up .Associations between each subscale of impulsivity and age at baseline and follow-up were tested. Increased attentional and motor subscales were positively correlated with age at baseline .There was no significant difference in the correlation between age and attentional subscale between baseline and follow-up  at baseline and in the SFG at follow-up . Change in motor subscale was negatively associated with FC in the SFG .Associations between each subscale of impulsivity and caudate-prefrontal cortex connectivity in the IFG and SFG at baseline and follow-up were tested. Motor subscale was negatively correlated with FC in the IFG .Associations between each subscale of impulsivity and BMI at baseline and follow-up were tested. A significant association was not observed (p < 0.0125).After the Bonferroni adjustment was performed for a multiple-comparison correction, the positive correlation between attentional impulsivity and age at baseline remained significant .There was no significant gender difference in any subscales . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YN and SK: conceptualization and design, reviewing and editing the manuscript. YN and SO: data acquisition. YN: data analysis and writing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of the present study was to evaluate the outcome of arthroscopic treatment for femoroacetabular impingement (FAI) syndrome 5 years post-surgery using patient-reported outcome scores (PROMs) validated for a young and active population with hip complaints.Patients were prospectively included in the study. A total of 184 patients , with mean age 38.0, underwent arthroscopic treatment for FAI syndrome and were analysed. Preoperatively and at the 5-year follow-up, patients completed a set of self-administered web-based PROMs consisting of the International Hip Outcome Tool (iHOT-12), the Copenhagen Hip and Groin Outcome Score (HAGOS), the Hip Sports Activity Scale (HSAS), the EuroQoL-5 Dimension Questionnaire (EQ-5D), the EQ-Visual Analogue Scale (VAS) and the VAS for overall hip function and overall satisfaction. The Wilcoxon signed rank test was used to compare preoperative PROM values with those obtained at the 5-year follow-up.p < 0.05), except for the HSAS score, which were unchanged; iHOT-12 (42.9 vs 67.2), HAGOS different subscales , EQ-5D (0.570 vs 0.742), EQ-VAS (66.6 vs 74.4), HSAS (3.13 vs 3.17) and VAS for overall hip function (47.9 vs 69.2). At the 5-year follow-up, 154 patients reported that they were satisfied with surgery (84.6%). Survivorship at the 5-year follow-up was 86.4%.A comparison of preoperative PROM scores and those obtained at the 5-year follow-up revealed statistically significant improvements for all outcome scores (Arthroscopic treatment for FAI syndrome yields good patient-reported outcome at the 5-year follow-up.II. Femoroacetabular impingement (FAI) syndrome is a cause of hip pain and reduced range of motion (ROM) in young and active patients. FAI syndrome has furthermore been proposed as a cause of osteoarthrosis (OA) of the hip –4. A proSurgical treatment for FAI syndrome aims to reproduce normal hip anatomy, thereby reducing pain and improving hip function. Several recent studies have reported good results following arthroscopic treatment for FAI syndrome at short- to mid-term follow-ups –11. The As previously noted , there iThe aim of the present study was to report outcomes at the 5-year follow-up after arthroscopic treatment for FAI syndrome, using patient-reported outcome measurements (PROM) developed for a young and active population. The primary hypothesis of the present study was that patients will experience a significant improvement at the 5-year follow-up.The study was conducted in agreement with the Helsinki Declaration. Informed consent was obtained from each patient in the study. Ethical approval for the study was granted by the Regional Ethical Review Board in Gothenburg at the Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden (registration number EPN 071-12).Patients treated between 2011 and 2013 were included in this prospective cohort study. The follow-up took place 5 years postoperatively. The inclusion criterion was arthroscopic surgery for FAI syndrome. The diagnosis of FAI syndrome was based on patient history, physical examination and radiological findings consistent with FAI syndrome. The indication for surgery was an established diagnosis of FAI syndrome and failed non-surgical treatment. The contraindications for surgery included advanced OA (joint space < 2 mm) and severe dysplasia . The exclusion criterion was prior hip surgery. All the hip arthroscopies were performed at two centres by three orthopaedic surgeons.There were 408 eligible patients, of which 47 patients were excluded due to prior hip surgery and 148 patients did not complete the 5-year follow-up. Twenty-nine patients (13.6%) converted to total hip arthroplasty during the follow-up period and were excluded. As a result, 184 patients were included for further analysis, 225 hips in total. The mean age was 38.0 (standard deviation (SD) ± 12.7) years and the youngest patient was 15 years of age. The demographic data are shown in Table Perioperative data were recorded at the time of surgery and included age, symptom duration, a description of cartilage status according to the classification by Konan et al.  and procAll the procedures were performed in an out-patient setting. The surgical technique has previously been described . An anteWith a clinically relevant change in iHOT-12 score of 10 points, a SD of 21 points , 2 were isolated pincer morphology (1.1%) and 104 had both cam and pincer morphology (57.8%). During the follow-up period, four re-operations were performed following index surgery (2.2%). The performed procedures are shown in Table .1%, 2 wep < 0.05), apart from the HSAS that was unchanged. Results and statistics are described in Table A comparison between preoperative PROM scores and those obtained at the 5-year follow-up revealed statistically significant improvements for all outcome scores  previously reported for the iHOT-12 and HAGOS, also indicating a clinically relevant improvement , 22. In The main strengths of the present study are the prospective study design, large patient group and long follow-up period. An additional strength is the use of multiple modern PROMs which have been validated for use in young and active patients. Despite good overall results, the HSAS level was more or less unchanged at follow-up. The reason why patients maintained the same level of physical activity even though their hip function improved may have many explanations, such as social factors and/or lifestyle changes. Another explanation could be that patients performed at a high level of physical activity prior to surgery, despite experiencing pain, which is common in athletes . SeveralThere are a number of limitations to the present study. As it is a cohort study, the lack of a control group makes it impossible to exclude any placebo effect on the results, or an effect independent of the treatment. However, long symptom duration prior to surgery should limit a return to the mean effect. Another limitation is the lack of recording of objective radiographic values, such as the alpha angle  for cam Arthroscopic treatment for FAI syndrome can be regarded as a viable treatment option, as promising PROM scores are reported at the 5-year follow-up; comparable to the results obtained at the 2-year follow-up. Future studies with longer outcomes including radiographic evaluation will also have the potential to reveal whether arthroscopic treatment for FAI syndrome has the potential to prevent the development of OA.